PROJECT SUMMARY Staphylococcus aureus is a Gram-positive opportunistic pathogen responsible for life-threatening infections in hospitals and communities alike. Especially concerning are methicillin-resistant S. aureus (MRSA) strains, which are resistant to ?-lactam antibiotics that target cell wall synthesis. Most MRSA strains also carry additional resistance markers rendering them resistant to multiple antibiotics, so treatment options are limited. Therefore, there is an urgent need to develop new antimicrobial therapies that are effective against S. aureus. Given that the cell envelope is the target of our first and best antimicrobials, studies aimed at understanding the mechanisms responsible for its assembly promise to uncover new vulnerabilities that can be targeted by future antimicrobial therapies. The proposed research will address two fundamental areas of S. aureus cell envelope assembly and morphogenesis. First, I address the mechanism by which methicillin-resistance factor PBP2a (a class b penicillin- binding protein, or bPBP) works with the rest of the cell wall synthesis machinery to promote ?-lactam resistance. ?-lactams like methicillin normally function by inhibiting the transpeptidase activity of bPBPs, which are essential for forming cell wall crosslinks and resisting turgor pressure. Recent research from my host laboratories and others has demonstrated that bPBPs act in complex with so-called ?separation, elongation, division, and sporulation? (SEDS) proteins, crosslinking new peptidoglycan polymerized by SEDS proteins into the growing cell wall. My preliminary results indicate that methicillin-insensitive PBP2a may function by replacing a methicillin- sensitive bPBP in a complex with the SEDS protein FtsW (the ?partner-swapping? hypothesis), restoring cell wall synthesis in the face of ?-lactam challenge. Second, I will design and employ high-throughput cytological screens to identify novel cell envelope biogenesis factors in S. aureus. In spite of the great importance and intensive study of S. aureus cell envelope, so far it has not been the subject of any such screen. Here, I utilize fluorescence- activated cell sorting (FACS) to screen a transposon library for envelope biogenesis defects, followed by deep sequencing of the transposon-genomic junctions of isolated mutants (Tn-seq). This approach has identified many novel mutants with an enhanced rate of cell lysis and with cell separation defects. Preliminary characterization suggests that these newly identified factors play roles in chromosome segregation, division site placement, and cell wall synthesis. The detailed characterization of many of these factors will likely continue beyond the period of this fellowship to form foundational projects in my own research laboratory, as well as provide potential targets for future small molecule antimicrobial therapies. The specific aims of this F32 application are to: AIM 1: Determine how PBP2a integrates into the native cell wall synthetic machinery. AIM 2: Identify novel factors that function in cell envelope biogenesis using cytological screens.